Increased osteoclastic resorption is responsible for multiple bone diseases, whether systemic (osteoporosis, Paget's disease, hyper-parathyroidism for instance) or local (rheumatoid arthritis, periodontal disease, multiple myeloma, tumor osteolysis etc.). Despite the existence of several anti- resorptive drugs, evidence of unwanted effects is emerging. It therefore remains of the utmost clinical importance to better understand the biology of the osteoclast (OC) and of bone resorption. Our laboratory has been heavily invested in the detailed molecular understanding of OC biology and bone resorption. One of the most salient features of this work, supported by many other laboratories, is the demonstration of the critical importance of the attachment of the OC to bone, mediated by a specialized adhesion structure, the actin-containing podosome, which in OCs is specifically organized in a peripheral belt (the actin ring) and ultimately in a sealing zone when OCs are on bone and actively resorbing. The formation of a podosome belt is an OC-specific feature. An important breakthrough has been the demonstration of the critical role played by microtubules (MTs) in the organization of podosomes into an actin ring and a sealing zone and consequently in bone resorption (Destaing et al. 2004; Gil-Henn et al., 2007; Purev et al., 2009). The primary goal of this application is to further elucidate the molecular mechanisms by which MTs are connected to and regulate the podosomes, and in particular the transition from clusters to a peripheral belt, and bone resorption. Our preliminary data revealed a functional interaction between MTs, the +Tip protein EB1, cortactin and podosomes, required for the formation of podosome belts. Our proposal focuses on the molecular regulation of the + ends of MTs and of their interaction with actin and actin-regulating proteins in podosomes in OCs. This will help understand the mechanisms that regulate the localization and turnover of podosomes and their organization in a peripheral belt, an OC-specific and necessary step in the establishment of the sealing zone, itself required for bone resorption. We propose to: 1- Determine the role of the +TIPs protein EB1 in linking MTs to actin dynamics in podosomes. 2- Determine the role of cortactin (and its close homolog HS1) and its interaction with EB1 in the link between MTs and actin dynamics in podosomes. 3- Analyze the role of Src and HDAC6, i.e. Tyrosine phosphorylation and acetylation, in the link between MTs and actin dynamics in podosomes. The studies may open new therapeutic avenues to regulate bone resorption, with potential impact on the treatment of osteoporosis, osteolytic diseases and also cancer, given the similarities between podosomes and invadopodia. PUBLIC HEALTH RELEVANCE: Increased osteoclastic resorption is responsible for multiple bone diseases, whether systemic (osteoporosis, Paget's disease, hyper-parathyroidism for instance) or local (rheumatoid arthritis, periodontal disease, multiple myeloma, primary or metastatic tumor osteolysis etc.). The primary goal of this application is to further elucidate the molecular mechanisms by which osteoclast attach to and resorb bone to better understand the biology of the osteoclast and of bone resorption to design new and safer drugs.